This P01 Program Project application seeks to develop insights into mechanisms by which antibodies (Abs) protect against HIV infection to facilitate design of improved Abs and effective immunogens. Development of effective vaccines or delivered Abs to control infection will require understanding of Ab interactions with antigen and with Ab receptors that mediate effector functions. Using knowledge of what Env mutations arise in response to HIV infection in humanized mice allows structural/bioinformatic analyses of which features promote Ab evasion, required information for designing broadly neutralizing antibodies (bNAbs) that are insensitive to common routes of viral evasion. This knowledge will allow optimization of the breadth/potency of bNAbs for passive delivery (both by injection and gene therapy reverse vaccination) and is required for effective immunogen design for vaccines, thus our project is relevant to both traditional and reverse vaccine strategies to combat HIV. To accomplish these goals and to establish basic principles underlying Ab-mediated protection, we will combine the expertise of the Nussenzweig, Ravetch, and Bjorkman laboratories in characterization of HIV bNAbs and humanized mouse models of HIV infection, antibody effector function evaluation and improvement, and the structural biology of Ab-HIV and Ab-receptor interactions. Our proposal comprises three separate, but inter-related and inter-dependent collaborative projects, with the following aims: (1) Test designed bNAbs in a humanized mouse model of HIV infection, sequence resistant HIV strains, evaluate bNAbs for ability to control established HIV infection in humanized mice, and evaluate novel immunogens in a mouse model; (2) Investigate the contributions of Fc effector function to HIV bNAbs in vitro and in vivo, including in a new in vivo mouse model for HIV entry and an AAV-based reverse immunization model in humanized mice; (3) Determine structural correlates of broad/potent neutralization and improved effector functions by solving crystal structures of designed and natural bNAbs complexed with HIV Env proteins and Fc receptors; design and test immunogens for eliciting bNAbs. These projects will be supported by an administrative core and three scientific cores comprising a cell/biochemical automation core to perform automated in vitro HIV neutralization and plate-binding assays, a protein expression core to express and purify recombinant proteins required for functional and structural studies, and an animal services core to generate/maintain mice required for in vivo experiments. RELEVANCE: HIV/AIDS remains a global epidemic with an urgent need for a vaccine and/or new therapies. Our project goals are to discover the mechanisms by which anti-HIV antibodies can prevent or treat infection (through Fab-mediated neutralization and Fc-mediated effector functions) and how HIV can escape through mutation, critical knowledge required for improving natural bNAbs as therapeutics and designing immunogens to elicit bNAbs. Project 1 - Human Antibodies to HIV Project Leader (PL): Nussenzweig, Michel DESCRIPTION (provided by applicant): The vast majority of HIV infected individuals develop antibodies to the virus. In most cases the antibodies only target the autologous strain, but some individuals develop neutralizing serologic responses to a broad range of different viral isolates. These responses are of interest because passive transfer of monoclonal antibodies with broad neutralizing activity to humanized mice or monkeys prevents infection. On the basis of these observations it has been proposed that a vaccine that elicits broadly neutralizing antibodies would be protective against HIV. However, little is known about the nature of the broadly neutralizing response. Only a small number of patients have been studied to date, and the majority of these patients are selected because their serologic activity focuses on the CD4 binding site of the viral envelope spike. The long-term goals of this proposal are to characterize new broadly neutralizing antibodies in terms of their functions in vivo and to understand how HIV-1 develops resistance to these antibodies in vivo. To accomplish these goals we propose three specific aims. First, we will develop an in vivo assay to assess the ability of broadly antibodies to prevent viral entry in mice. HIV neutralization is currently assayed in vitro using TZM-bl cells. The new mouse model will be used to examine the relative efficacy of different monoclonal antibodies and the contribution of innate effector mechanisms to blocking HIV entry in vivo. Second, we will define the basis for development of resistance to broadly neutralizing antibodies in HIV infected humanized mice. We will use the information for structure based rational design approaches to iteratively enhance antibody breadth and potency. The ultimate goal of this part of the proposal is to determine which of the large group of currently available antibodies might be most useful for passive vaccine and immunogen design approaches. Finally, we will evaluate potential immunogens designed using structural and other data in knock-in mice containing human germline antibody precursor genes. Taken together, these experiments should help inform future clinical studies in which neutralizing antibodies might be considered for use in passive therapy or prevention studies and which of their targets would be most useful for immunization strategies. RELEVANCE: Although there is still no vaccine for HIV, a small number of infected individuals develop antibodies that can prevent the infection. The proposed research aims to develop an understanding of these antibodies with the long term goal of being able to elicit them de novo as a component of a vaccine to be used in un-infected individuals.